Modulation of LMNA splicing as a strategy to treat prelamin A diseases
John M. Lee, … , Stephen G. Young, Loren G. Fong
John M. Lee, … , Stephen G. Young, Loren G. Fong
Published April 1, 2016; First published March 21, 2016
Citation Information: J Clin Invest. 2016;126(4):1592-1602. https://doi.org/10.1172/JCI85908.
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Research Article Aging Therapeutics

Modulation of LMNA splicing as a strategy to treat prelamin A diseases

Abstract

The alternatively spliced products of LMNA, lamin C and prelamin A (the precursor to lamin A), are produced in similar amounts in most tissues and have largely redundant functions. This redundancy suggests that diseases, such as Hutchinson-Gilford progeria syndrome (HGPS), that are caused by prelamin A–specific mutations could be treated by shifting the output of LMNA more toward lamin C. Here, we investigated mechanisms that regulate LMNA mRNA alternative splicing and assessed the feasibility of reducing prelamin A expression in vivo. We identified an exon 11 antisense oligonucleotide (ASO) that increased lamin C production at the expense of prelamin A when transfected into mouse and human fibroblasts. The same ASO also reduced the expression of progerin, the mutant prelamin A protein in HGPS, in fibroblasts derived from patients with HGPS. Mechanistic studies revealed that the exon 11 sequences contain binding sites for serine/arginine-rich splicing factor 2 (SRSF2), and SRSF2 knockdown lowered lamin A production in cells and in murine tissues. Moreover, administration of the exon 11 ASO reduced lamin A expression in wild-type mice and progerin expression in an HGPS mouse model. Together, these studies identify ASO-mediated reduction of prelamin A as a potential strategy to treat prelamin A–specific diseases.

Authors

John M. Lee, Chika Nobumori, Yiping Tu, Catherine Choi, Shao H. Yang, Hea-Jin Jung, Timothy A. Vickers, Frank Rigo, C. Frank Bennett, Stephen G. Young, Loren G. Fong

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Figure 1

Identification of a region within exon 11 of Lmna that affects prelamin A and lamin C expression.

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Identification of a region within exon 11 of Lmna that affects prelamin ...
(A) qRT-PCR identifies several ASOs that increase the lamin C/prelamin A ratio. Wild-type MEFs were transfected with overlapping 18-nt ASOs corresponding to sequences in exon 11 of Lmna. After 2 days, mRNA levels for prelamin A, lamin C, and total lamin A/C were measured by qRT-PCR. Total lamin A/C levels (black bars) and the lamin C/prelamin A ratios (white bars) were calculated and expressed relative to control-treated cells (Con), set at a value of 1.0. (B) ASO E1-31 promotes lamin C splicing. Wild-type MEFs were transfected with ASOs E11-26, E11-31, E11-36, and I10-11 (I10-11 is located in lamin C’s 3′ UTR). After 2 days, mRNA levels for prelamin A and lamin C were measured by qRT-PCR. The expression was normalized to levels in cells that were treated with transfection reagent alone (nontemplate control [NTC]) (set at a value of 1.0). The mean ± SEM for 3 independent experiments is shown. Comparison with nontemplate control: *P < 0.05; **P < 0.01, t test. (C) Western blots showing that ASOs modulate lamin A and lamin C protein expression. MEFs were transfected in duplicate with ASOs I11-146, I10-11, E11-26, E11-31, and E11-36. After 2 days, cell extracts were prepared and analyzed by Western blotting with antibodies against lamin A/C and actin. The band intensities were measured by infrared laser scanning and normalized to actin. The results were expressed relative to the nontemplate control, set at a value of 1.0.